1. Field of the Invention
The present invention relates to methods and systems for microfluidic DNA sample preparation. More specifically, embodiments of the present invention relate to methods and systems for the isolation of DNA from patient samples on a microfluidic device and use of the DNA for downstream processing, such as performing amplification reactions and thermal melt analysis on the microfluidic device.
2. Description of Related Art
The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase chain reaction (PCR) is perhaps the most well-known of a number of different amplification techniques.
PCR is one of the more sensitive methods for nucleic acid analysis. However, many substances in clinical samples, including blood, can affect PCR and can result in substantial error in the PCR results. Thus, DNA isolation and purification are critical to methods for DNA analysis. Conventional DNA preparation requires large volume samples and requires a long process time. Microfluidic technology makes it possible to use much less sample and less time for DNA sample preparation. Solid phase extraction methods have been applied in DNA sample preparation. DNA is selectively extracted on the solid phase while other substances in the sample are washed out of the extraction column. For instance, Breadmore et al. (Anal Chem 75(8):1880-1886, 2003) reported on a microchip-based DNA purification method using silica beads packed into glass microchips and immobilized within a sol-gel. Alternatively, DNA isolation can be achieved by nuclei size sieving.
Since DNA only exists in the nuclei of cells, DNA samples can be prepared by selectively isolating nuclei from the sample. Traditional nuclei isolation is slow and has low efficiency. Generally, nuclei isolation is performed by selective lysis of cellular membranes while keeping the nuclei intact. Nuclei are then isolated by centrifuge, sediment or sieving. Dignani et al. (Nucl Acids Res 11:1475-1489, 1983) reported isolation of nuclei from samples by centrifugation. U.S. Pat. No. 5,447,864 discloses a method of isolating nuclei using a DNA mesh. U.S. Pat. No. 6,852,851 discloses a method of isolating nuclei in a microfabricated apparatus that contains a plurality of radially dispersed micro-channels. U.S. Pat. No. 6,992,181 describes the use of a CD device for the purification of DNA or cell nuclei. This method requires moving parts and centrifugal force to isolate DNA and or cell nuclei, using a barrier in the channel to impede flow of DNA and nuclei. Palaniappan et al. (Anal Chem 76:6247-6253, 2004) reported a continuous flow microfluidic device for rapid erythrocyte lysis. VanDelinder et al. (Anal Chem 78:3765-3771, 2006) reported a separation of plasma from whole human blood in a continuous cross-flow in a molded microfluidic device. To increase mixing of lysis buffer with blood sample in microfluidic channel, Palaniappan et al. (Anal Chem 78:5453-5461, 2006) reported a microfluidic channel with the channel floors that are patterned with double herringbone microridges. VanDelinder et al. (Anal Chem 79:2023-2030, 2007) describe a perfusion in microfluidic cross-flow for particles and cells. Particles flow in the main channel while a perfusion flows through the side channels to exchange the medium of suspension.
There are several problems with current technology of purifying DNA by isolating nuclei from cells. First, the conventional approach is slow. Usually, the conventional approach takes hours to finish from cell lysis to the nuclei isolation. For example, the purification process described in U.S. Pat. No. 6,852,851 is carried out in a plurality of micro channels with a mesh built into the micro channel. However, because the size of micro channels is limited, the process can treat only limited sample sizes from 100 nl to 1 μl. Another problem is with the method of releasing DNA and/or nuclei from membrane. For example, DNAse is used in U.S. Pat. No. 5,447,864 to release nuclei from membrane. However, the addition of DNAse will fail the down stream process. In U.S. Pat. No. 5,447,864, sodium dodecyl sulfate solution or proteinase K is used to disrupt the nuclear envelope in order to release DNA. However, these lysis reagents will also seriously inhibit the downstream process. The conventional nuclear lysis method is to use high concentration sodium chloride (0.5 M) to disrupt the nuclear membrane. However, the high concentration sodium chloride will also inhibit the downstream process.
In addition, the current technologies require specific buffers for DNA binding and washing, most of which are not compatible with down stream applications such as PCR. These technologies also have a wide range of efficiencies in the overall quantity of DNA that is purified. This can be a significant problem when samples are to be used in microfluidics. The multiple reagents that are typically required for DNA purification would demand that moving parts, such as valves, be constructed into a microfluidic device for the introduction of multiple reagents in a solid phase extraction. In a microfluidic system, solid phase extraction or the use of multiple reagents is complicated and can lead to system failures.
Although the various methods exist to capture nuclei for use in down stream application or to separate specific cells from a sample population, none of these methods describes a single device that is capable of extracting cell nuclei and isolating the nucleic acid contained in the cell nucleic that is suitable for microfluidic processing and down stream processes such as amplification reactions and detection analysis. Thus, there is a need to develop microfluidic systems and methods for DNA isolation.